The cellular complexity of the nervous system sets it apart from other tissues. Asymmetric cell divisions, in which a precursor cell divides to produce two sibling cells of different fates, are central to the generation of cell diversity in nervous systems. Most asymmetric divisions in Drosophila and vertebrate nervous systems depend on the opposing activities of the Notch signaling pathway and the cytoplasmic determinant Numb. During precursor divisions Numb segregates exclusively into one sibling cell where it blocks Notch signaling to prevent adoption of the Notch-dependent fate. The absence of Numb in the other sibling allows Notch signaling, and thus, adoption of the Notch-dependent fate. Present models suggest that Numb blocks Notch activity by promoting endocytosis of the Notch receptor. However, there are significant caveats to this model. For example, both sibling cells exhibit equivalent levels of Notch at the cell membrane and a truncated form of Numb deleted for all known endocytic motifs is functional during Notch-mediated asymmetric divisions. Work from our lab on Sanpodo, a novel transmembrane protein required for Notch signaling only during asymmetric divisions, suggests a different model. Sanpodo localizes to the cell membrane of the sibling cell whose fate depends on Notch activity, while in the other cell Numb blocks Sanpodo from localizing to the cell membrane. These observations led to the model that Sanpodo acts at the cell membrane to promote Notch signaling, and that Numb blocks Notch activity by keeping Sanpodo off of the cell membrane. However, the molecular mechanisms by which Sanpodo promotes Notch activity arid Numb regulates Sanpodo localization remain unknown. This proposal seeks to elucidate the molecular basis of Sanpodo function and regulation. Specifically, we propose to (i) identify the functional domains of Sanpodo via structure/function studies, (ii) identify and characterize factors that interact genetically or physically with sanpodo via complementary biochemical and genetic approaches and (iii) search computationally for vertebrate sanpodo genes. Defects in Notch activity are being implicated in a growing number of diseases, including multiple types of brain cancer. And while no homolog of Sanpodo has yet been identified in vertebrates, based on (i) the high degree of divergence between the amino acid sequences of Sanpodo in insects and (ii) the conservation of the Notch/numb molecular machinery as a fundamental mechanism regulating asymmetric divisions, we hypothesize the existence of a functional Sanpodo homolog with limited primary sequence homology. Thus, identifying vertebrate sanpodo genes and elucidating the molecular basis of Sanpodo function and regulation will provide key insight into the molecular control of asymmetric divisions. Such insight should help us understand the etiology of diseases in which this process is de-regulated and design new methods to treat these diseases.